Pharmaceutical composition comprising Lefty

ABSTRACT

The present disclosure relates to suppression of cellular transformation and dysplasia by topical application of Lefty. In particular, the present disclosure provides compositions and methods for topically applying Lefty to treat and prevent cancers.

The present application is a divisional of U.S. application Ser. No.15/029,829, filed Apr. 15, 2016, which is a 371 U.S. National PhaseEntry of International Application No. PCT/US2014/061048, filed Oct. 17,2014, which claims priority to U.S. Patent Application Ser. No.61/892,076, filed Oct. 17, 2013, the disclosures of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present disclosure relates to suppression of cellular transformationand dysplasia by topical application of Lefty. In particular, thepresent disclosure provides compositions and methods for topicallyapplying Lefty to treat and prevent disease.

BACKGROUND OF THE INVENTION

Melanoma is a malignant tumor of melanocytes. Melanocytes produce thedark pigment, melanin, which is responsible for the color of skin. Thesecells predominantly occur in skin, but are also found in other parts ofthe body, including the bowel and the eye (uveal melanoma). Melanoma canoriginate in any part of the body that contains melanocytes.

Melanoma is less common than other skin cancers. However, it is muchmore dangerous if it is not found early. It causes the majority (75%) ofdeaths related to skin cancer. Worldwide, doctors diagnose about 160,000new cases of melanoma yearly. In women, the most common site is the legsand melanomas in men are most common on the back. It is particularlycommon among Caucasians, especially northwestern Europeans living insunny climates. There are high rates of incidence in Oceania, NorthernAmerica, Europe, Southern Africa, and Latin America, with a paradoxicaldecrease in southern Italy and Sicily. This geographic pattern reflectsthe primary cause, ultraviolet light (UV) exposure crossed with theamount of skin pigmentation in the population.

According to a WHO report, about 48,000 melanoma related deaths occurworldwide per year. The treatment includes surgical removal of thetumor. If melanoma is found early, while it is still small and thin, andif it is completely removed, then the chance of cure is high. Thelikelihood of the melanoma coming back or spreading depends on howdeeply it has gone into the layers of the skin. For melanomas that comeback or spread, treatments include chemo- and immunotherapy, orradiation therapy.

Additional methods for treating and preventing melanoma are needed.

SUMMARY OF THE INVENTION

The present disclosure relates to suppression of cellular transformationand dysplasia by topical application of Lefty. In particular, thepresent disclosure provides compositions and methods for topicallyapplying Lefty to treat and prevent disease.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition for topical administration comprising a Lefty polypeptide.In some embodiments, the Lefty is glycosylated. In some embodiments, theLefty is recombinant human Lefty or is isolated from human embryonicstem cells. In some embodiments, the composition comprises histamine. Insome embodiments, the composition comprises one or more additionalcomponents (e.g., ethanol, trolamine, carbomer, methyl paraben, propylparaben, or PBS). In some embodiments, Lefty is present in saidcomposition as a concentration of between 1 ng/ml and 1000 ng/ml (e.g.,between 10 and 500 ng/ml).

The present disclosure further provides a method of treating orpreventing a disease on a surface of the body of a subject, comprisingcontacting the surface with any one of the topical Lefty compositionsdescribed herein. In some embodiments, the disease is cancer, forexample, a skin cancer (e.g., basal cell carcinoma, squamous cellcarcinoma, or malignant melanoma). In some embodiments, the surface is,for example, a nevi, a site comprising a pre-cancerous lesion, a tissueof ectodermal origin (e.g., epidermis, mucosal surfaces, cornea, neuralcrest origin tissue, melanocytes, or retina), the site of removal of acancerous or precancerous lesion, or a site suspected of comprising acancerous or pre-cancerous lesion. In some embodiments, the contactingtreats cancer or prevents a pre-cancerous lesion from becomingcancerous. In some embodiments, the contacting is performed continuouslyover a period of weeks or months (e.g., one or more times a day for aperiod of days, weeks, months, or years).

In some embodiments, the method further comprises the step ofadministering a known chemotherapy agent. In some embodiments, the knownchemotherapy agent is administered concurrently or sequentially with thetopical Lefty composition. For example, in some embodiments, the knownchemotherapeutic agent is administered prior to or following the topicalLefty composition. In some embodiments, there is gap of several days(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days) between theadministration of the topical Lefty composition and the administrationof the known chemotherapeutic agent.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc.

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,recombinant polypeptides are expressed in bacterial host cells and thepolypeptides are purified by the removal of host cell proteins; thepercent of recombinant polypeptides is thereby increased in the sample.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Such examples are nothowever to be construed as limiting the sample types applicable to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to suppression of cellular transformationand dysplasia by topical application of Lefty. In particular, thepresent disclosure provides compositions and methods for topicallyapplying Lefty to treat and prevent diseases associated with cellulartransformation or dysplasia.

I. Pharmaceutical Compositions Comprising Lefty

Embodiments of the present disclosure provide compositions and methodsfor topical application of Lefty. The present disclosure is not limitedto particular Lefty polypeptides or topical formulations. ExemplaryLefty polypeptides and formulations are described herein.

A. Lefty Polypeptides

The present disclosure is not limited to a particular Lefty polypeptide.Exemplary Lefty polypeptides are described, for example, in U.S. Pat.No. 8,106,004, herein incorporated by reference in its entirety. As usedherein, the terms “Lefty A/B” and “Lefty” are interchangeable and referto either Lefty A or Lefty B, or both Lefty A and Lefty B incombination. In some embodiments, Lefty is isolated from amicroenvironment (e.g., an environment that comprises a basementmembrane or other defined matrix that is in contact with embryonic stemcells, such as human embryonic stem cells (hESCs)). In one embodiment,Lefty, isolated from a microenvironment, may be substantially pure. Inanother embodiment, Lefty may be present in combination with other hESCfactors. As used herein, by “substantially pure” it is meant apreparation which is at least 70% by weight (dry weight) the compound ofinterest, e.g., hESC-derived Lefty. Preferably the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. Purity can be measured by anyappropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

In another embodiment, the invention provides an isolated Lefty proteinproduced by conditioning a matrix with human embryonic stem cells. Asused herein, “conditioning a matrix” refers to preparing apreconditioned microenvironment as defined herein. In certainembodiments, the matrix is conditioned with hESCs from 0 to 10 days orany range in between, including, but not limited to, from 0.5 to 10days, from 2 to 8 days, from 3 to 6 days, from 3 to 5 days, from 3 to 4days, or for 1, 2, 3, 4, or 5 days. Lefty may be isolated from thematrix by any method known to one of skill in the art, including throughuse of anti-Lefty antibodies.

In one embodiment, the invention provides a protein comprisingglycosylated Lefty. In this embodiment, Lefty may be glycosylated tovarying degrees, and may comprise one or more N- and/or O-linkedglycosylation sites, or a combination thereof. In one embodiment, theglycosylated Lefty is characterized in that more than 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the possible N- and/or O-glycosylation sites areglycosylated. In another embodiment, the glycosylated Lefty ischaracterized in that less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of thepossible N- and/or O-glycosylation sites are glycosylated. In anotherembodiment, the glycosylated Lefty is characterized in that thepercentage of possible N- and/or O-glycosylation sites that areglycosylated is based on a combination of the “more than” and “lessthan” percentages recited above. Thus, in one non-limiting example, theglycosylated Lefty is characterized in that more than 30% and less than70% of the possible N- and/or O-glycosylation sites are glycosylated. Inanother embodiment, 100% of the possible N- and/or O-glycosylation sitesare glycosylated.

In one embodiment, the glycosylated Lefty is glycosylated tosubstantially the same extent as Lefty derived from hESCs.

Glycosylated Lefty may be prepared by any method, including byrecombinant methods (see, e.g. Sambrook et al., 2001, Molecular Cloning:A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). In one embodiment, glycosylated Lefty is preparedrecombinantly in Chinese Hamster Ovary (CHO) or human embryonic kidney(HEK) cells. Alternatively, glycosylated Lefty may be prepared bychemical synthesis methods (such as solid phase peptide synthesis) usingtechniques known in the art such as those set forth by Merrifield etal., 1963, J. Am. Chem. Soc. 85:2149; Houghten et al., 1985, Proc NatlAcad. Sci. USA 82:5132; and Stewart and Young, Solid Phase PeptideSynthesis (Pierce Chemical Co. 1984), or by a combination of syntheticand recombinant techniques. Glycosylated Lefty may also be prepared byisolation from hESCs, including by isolation from the microenvironmentof hESCs.

Included within the scope of the invention are fragments or derivativesof Lefty or glycosylated Lefty. As used herein, “fragment” means anyportion of the full length Lefty sequence having an activity of the fulllength protein, including, but not limited to, the ability to inhibitNodal. Included in the scope of “fragments” are naturally occurringenzymatic cleavage products. Included in the scope of the term“derivatives” are derivatives of full length Lefty as well as fragmentsthereof. As used herein, “derivative” or “derivatives” includesvariations of Lefty having one or more amino acid residues which havebeen added, deleted, inserted or substituted, where the resultingpolypeptide has an activity of Lefty, including, but not limited to, theability to inhibit Nodal. As used herein, “derivatives” also includeschemical derivatives of Lefty and variations thereof. It will beunderstood to one of skill in the art that these variations may occur inany combination.

In some embodiments the Lefty compositions comprise a Lefty peptidemimetic (peptidomimetic). The use of peptides as lead compounds, andsubsequently conversion into low-molecular-weight nonpeptide molecules(peptidomimetics), has led to development of small-molecule antagonistsof intracellular targets (Bottger et al., J Mol Biol, 1997. 269(5): p.744-56; Bottger et al., Oncogene, 1996. 13(10): p. 2141-7). Therefore,peptidomimetics have emerged as a powerful means for overcoming thelimitations inherent in the physical characteristics of peptides,improving their therapeutic potential (Kieber-Emmons et al., Curr OpinBiotechnol, 1997. 8(4): p. 435-41; Beeley, Trends Biotechnol, 1994.12(6): p. 213-6; Moore et al., Trends Pharmacol Sci, 1994. 15(4): p.124-9). In some embodiments, compared to native peptides,peptidomimetics possess desirable pharmacodynamic properties superior tonatural peptides, including good oral activity, long duration of action,better transport through cellular membranes, decreased rate ofexcretion, and decreased hydrolysis by peptidases.

Development of a small molecule peptidomimetic generally involvesidentification of the smallest functional peptide unit capable ofinhibiting the targeted interaction. A growing body of literaturedemonstrates that high-affinity ligands can be selected from peptidelibraries displayed on bacteriophages (Sulochana and Ge, Curr Pharm Des,2007. 13(20): p. 2074-86; Cwirla et al., Proc Natl Acad Sci USA, 1990.87(16): p. 6378-82; Scott and Smith, Science, 1990. 249(4967): p.386-90; Devlin et al., Science, 1990. 249(4967): p. 404-6), and manyapplications have been directed toward antagonizing the function of aprotein ligand (Dower, Curr Opin Chem Biol, 1998. 2(3): p. 328-34; Sidhuet al., Methods Enzymol, 2000. 328: p. 333-63). Because the librariescan be very large (10¹¹ or more individual members), no initialassumptions are required concerning how to bias the library, nor theselective enrichment of rare binding phage through biologicalamplification and rescreening. Those sequences that bind can beidentified by sequencing their encoding DNA.

Chemically modified derivatives of glycosylated Lefty may be prepared byone skilled in the art, in view of the disclosures described herein.Glycosylated Lefty derivatives are modified in a manner that isdifferent—either in the type or location of the molecules naturallyattached to the polypeptide. Derivatives may include molecules formed bythe deletion of one or more naturally-attached chemical group, or theymay be modified by the covalent attachment of one or more polymers. Forexample, the polymer selected is typically water-soluble so that theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. Included within thescope of suitable polymers is a mixture of polymers. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water-soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is preferably between about 5 kDa andabout 50 kDa, more preferably between about 12 kDa and about 40 kDa andmost preferably between about 20 kDa and about 35 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C.sub.1-C.sub.10), alkoxy-,or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol,dextran (such as low molecular weight dextran of, for example, about 6kD), cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols(e.g., glycerol), and polyvinyl alcohol. Also encompassed by the presentinvention are bifunctional crosslinking molecules which may be used toprepare covalently attached glycosylated Lefty polypeptide multimers.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.The optimal reaction conditions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofpolymer molecules to protein, the greater the percentage of attachedpolymer molecule. In one embodiment, the glycosylated Lefty derivativemay have a single polymer molecule moiety at the amino-terminus. See,e.g., U.S. Pat. No. 5,234,784.

The pegylation of a polypeptide may be specifically carried out usingany of the pegylation reactions known in the art. Such reactions aredescribed, for example, in the following references: Francis et al.,1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and0401384; and U.S. Pat. No. 4,179,337.

In another embodiment, glycosylated Lefty polypeptides may be chemicallycoupled to biotin. The biotin/glycosylated Lefty polypeptide moleculesare then allowed to bind to avidin, resulting in tetravalentavidin/biotin/glycosylated Lefty polypeptide molecules. GlycosylatedLefty polypeptides may also be covalently coupled to dinitrophenol (DNP)or trinitrophenol (TNP) and the resulting conjugates precipitated withanti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of10.

Generally, conditions that may be alleviated or modulated by theadministration of the present glycosylated Lefty derivatives includethose described herein for glycosylated Lefty. However, the glycosylatedLefty derivatives disclosed herein may have additional activities,enhanced or reduced biological activity, or other characteristics, suchas increased or decreased half-life, as compared to the non-derivatizedmolecules.

B. Compositions for Topical Application

In some embodiments, the present invention provides compositionscomprising Lefty that are formulated for topical administration. In someembodiments, pharmaceutical compositions comprise an effective amount ofone or a plurality of Nodal inhibitors together with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservativeand/or adjuvant, wherein the pharmaceutical composition is capable ofinducing a desired therapeutic effect when properly administered to apatient. Preferably, acceptable formulation materials are nontoxic torecipients at the dosages and concentrations employed.

In some embodiments, topical formulations are formulated to preventdegradations of Lefty and allow the polypeptide to enter skin cells. Insome embodiments, topical formulations comprise one or more of thefollowing components: histamine dihydrochloride, ethanol, trolamine,carbomer (e.g., Carbopol 974P), methyl paraben, propyl paraben, Lefty(e.g., rhLefty), and PBS. In some embodiments, compositions for topicaladministration are aqueous and do not include any waxy components. Insome embodiments, compositions are formulated as liposomal formulations.In some embodiments, topical compositions are formulated for spray oraerosol applications.

In some embodiments, topical formulations for ophthalmic use areformulated (e.g., as saline drops).

In some embodiments, compositions comprise Lefty (e.g., a glycosylatedLefty) at a dosage between 0.01 and 500 ng/mL, between 0.01 and 200ng/mL, between 0.1 and 200 ng/mL, between 0.1 and 100 ng/mL, between 1and 100 ng/mL, between 10 and 100 ng/mL, between 10 and 75 ng/mL,between 20 and 75 ng/mL, between 20 and 50 ng/mL, between 25 and 50ng/mL, or between 30 and 40 ng/mL.

In certain embodiments, a pharmaceutical composition useful in themethods of the invention may contain formulation materials formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.In such embodiments, suitable formulation materials include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine,arginine or lysine); antimicrobials; antioxidants (such as ascorbicacid, sodium sulfite or sodium hydrogen-sulfite); buffers (such asborate, bicarbonate, Tris-HCl, citrates, phosphates or other organicacids); bulking agents (such as mannitol or glycine); chelating agents(such as ethylenediamine tetraacetic acid (EDTA)); complexing agents(such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. SeeREMINGTON'S PHARMACEUTICAL SCIENCES, 18.sup.th Edition, (A. R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical compositions aredetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of release and rate of clearance of the Lefty.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water,possibly supplemented with other materials common in compositions fortopical administration.

Alternatively, it is possible to entrap the therapeutic agents, fordelivery, in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example, by the use ofhydroxymethyl cellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin, microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences (1980),herein incorporated by reference.

The compounds of this invention can also be administered by atransdermal device. Preferably topical administration will beaccomplished using a patch either of the reservoir and porous membranetype or of a solid matrix variety. In either case, the active agent isdelivered continuously from the reservoir or microcapsules through amembrane into the active agent permeable adhesive, which is in contactwith the skin or mucosa of the recipient. If the active agent isabsorbed through the skin, a controlled and predetermined flow of theactive agent is administered to the recipient. In the case ofmicrocapsules, the encapsulating agent may also function as themembrane. The transdermal patch may include the compound in a suitablesolvent system with an adhesive system, such as an acrylic emulsion, anda polyester patch. All transdermal delivery systems are contemplatedherein, including, but not limited to, single-layer drug-in-adhesives,multi-layer drug-in-adhesives, reservoirs, matrixes, vapour patches,wafers (e.g., lyophilized wafers), systems employing iontophoresis,non-cavitational ultrasound, electroporation, cavitational ultrasound,microneedles, thermal ablation, microdermabrasion, and the like.

II. Uses of Topical Lefty Formulations

Embodiments of the present disclosure provide topical Lefty compositionsfor treating and preventing disease on body surfaces or mucosal layers.In some embodiments, topical Lefty formulations find use for applicationto any tissue of ectodermal original (e.g., skin, mucosal surfaces,cornea, neural crest origin tissue (e.g., melanocytes, retina, etc.),etc.). The present disclosure is not limited to a particular cancer.Topical Lefty formulations find use in the treatment of variety ofcancers and precancerous conditions. Examples include, but are notlimited to, skin cancer (e.g., basal cell carcinoma (BCC), squamous cellcarcinoma (SCC) and malignant melanoma), extodermal dysplasia, and othercancers of body or mucosal (e.g., buccal mucosa, esophageal mucosa,gastric mucosa, intestinal mucosa. nasal mucosa, olfactory mucosa, oralmucosa, bronchial mucosa, endometrium, or penile mucosa).

In some embodiments, topical Lefty compositions are used to treatcancers. For example, in some embodiments, a topical Lefty compositionis applied to a cancerous lesion. In some embodiments, a topical Leftycomposition is applied to a removal site after surgical or other removalof a cancerous lesion.

In some embodiments, topical Lefty compositions are used to treatpre-cancerous conditions or prevent pre-cancerous lesions fromprogressing to cancers. For example, in some embodiments, a topicalLefty composition is applied to a pre-cancerous lesion. In someembodiments, a topical Lefty composition is applied to a removal siteafter surgical or other removal of a pre-cancerous lesion.

In some embodiments, the cancerous or pre-cancerous lesion is a nevi.For example, in some embodiments, nevi suspected of being cancerous orpre-cancerous or diagnosed as cancerous or pre-cancerous are treatedwith a topical Lefty composition. In some embodiments, the removal siteof a nevi is treated with a topical Lefty composition after the nevi isremoved.

In some embodiments, the nevi or subjects identified or suspected ofhaving a high risk of developing cancerous nevi are prophylacticallytreated with a topical Lefty composition.

The present disclosed is not limited to the treatment and prevention ofcancer. In some embodiments, topical Lefty compositions find use thetreatment of artificial skin or skin transplants derived from stemcells. In such embodiments, the topical Lefty compositions preventfurther differentiation of the skin cells (e.g., to control modeling andarchitecture).

In some embodiments, topical Lefty compositions are applied to the eye.In some embodiments of such ophthalmic applications, the Leftyformulation is typically in a saline drop form. In some embodiments,topical Lefty compositions are used to treat disorders of the eyemucosal membranes such as cancers and other diseases.

In some embodiments, topical Lefty compositions are used to treat cancerstem cells (e.g., by preventing differentiation into cancers).

In some embodiments, treatment with topical Lefty compositions isadministered one or more times to a given location (e.g., one or moretimes a day for a period of days, weeks, or months). In someembodiments, treatment is long term or ongoing (e.g., administered oneor more times per day for a period of weeks, months, or years).

Administration may be conducted by any suitable method, including, butnot limited to, application of a transdermal device, irrigation of alesion location or site of removal of a lesion, spray delivery, dropletdelivery, nebulizer delivery, pump delivery, powder delivery, geldelivery, and the like.

III. Combination Therapy

In some embodiments, the present invention provides therapeutic methodscomprising one or more compositions described herein in combination withan additional agent (e.g., a chemotherapeutic agent). The presentinvention is not limited to a particular chemotherapy agent.

In some embodiments, a topical Lefty composition is administeredconcurrently with an additional agent (e.g., chemotherapy agent). Insome embodiments, a topical Lefty composition and an additional agentare administered sequentially. For example, in some embodiments, atopical Lefty composition is administered following an additional agent.In some embodiments, there is a gap (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more day or several weeks or months) in between administration of thetopical Lefty composition and the additional agent.

Various classes of antineoplastic (e.g., anticancer) agents arecontemplated for use in certain embodiments of the present invention.Anticancer agents suitable for use with embodiments of the presentinvention include, but are not limited to, agents that induce apoptosis,agents that inhibit adenosine deaminase function, inhibit pyrimidinebiosynthesis, inhibit purine ring biosynthesis, inhibit nucleotideinterconversions, inhibit ribonucleotide reductase, inhibit thymidinemonophosphate (TMP) synthesis, inhibit dihydrofolate reduction, inhibitDNA synthesis, form adducts with DNA, damage DNA, inhibit DNA repair,intercalate with DNA, deaminate asparagines, inhibit RNA synthesis,inhibit protein synthesis or stability, inhibit microtubule synthesis orfunction, and the like.

In some embodiments, exemplary anticancer agents suitable for use incompositions and methods of embodiments of the present inventioninclude, but are not limited to: 1) alkaloids, including microtubuleinhibitors (e.g., vincristine, vinblastine, and vindesine, etc.),microtubule stabilizers (e.g., paclitaxel (TAXOL), and docetaxel, etc.),and chromatin function inhibitors, including topoisomerase inhibitors,such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide(VM-26), etc.), and agents that target topoisomerase I (e.g.,camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-bindingagents (alkylating agents), including nitrogen mustards (e.g.,mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, andbusulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine,and semustine, etc.), and other alkylating agents (e.g., dacarbazine,hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalentDNA-binding agents (antitumor antibiotics), including nucleic acidinhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines(e.g., daunorubicin (daunomycin, and cerubidine), doxorubicin(adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g.,anthracycline analogues, such as mitoxantrone, etc.), bleomycins(BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4)antimetabolites, including antifolates (e.g., methotrexate, FOLEX, andMEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP,PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir,chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g.,fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine(FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR(ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase,and hydroxyurea, etc.; 6) hormones, including glucocorticoids,antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g.,flutamide, etc.), and aromatase inhibitors (e.g., anastrozole(ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin andcarboplatin, etc.); 8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; 9) biological responsemodifiers (e.g., interferons (e.g., IFN-α, etc.) and interleukins (e.g.,IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growthfactors; 12) agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14)antisense therapy techniques; 15) tumor vaccines; 16) therapies directedagainst tumor metastases (e.g., batimastat, etc.); 17) angiogenesisinhibitors; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors ofacetylation and/or methylation (e.g., HDAC inhibitors); 20) modulatorsof NF kappa B; 21) inhibitors of cell cycle regulation (e.g., CDKinhibitors); 22) modulators of p53 protein function; and 23) radiation.

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of embodiments of the presentinvention. For example, the U.S. Food and Drug Administration maintainsa formulary of oncolytic agents approved for use in the United States.International counterpart agencies to the U.S. F.D.A. maintain similarformularies. The below Table provides a list of exemplary antineoplasticagents approved for use in the U.S. Those skilled in the art willappreciate that the “product labels” required on all U.S. approvedchemotherapeutics describe approved indications, dosing information,toxicity data, and the like, for the exemplary agents.

Aldesleukin Proleukin Chiron Corp., (des-alanyl-1, serine-125 humanEmeryville, CA interleukin-2) Alemtuzumab Campath Millennium and (IgG1κanti CD52 antibody) ILEX Partners, LP, Cambridge, MA AlitretinoinPanretin Ligand (9-cis-retinoic acid) Pharmaceuticals, Inc., San DiegoCA Allopurinol Zyloprim GlaxoSmithKline, (1,5-dihydro-4 H-pyrazolo[3,4-Research Triangle d]pyrimidin-4-one monosodium salt) Park, NCAltretamine Hexalen US Bioscience, (N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-West triazine-2,4,6-triamine) Conshohocken, PA Amifostine Ethyol USBioscience (ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen phosphate(ester)) Anastrozole Arimidex AstraZeneca(1,3-Benzenediacetonitrile,a,a,a′,a′- Pharmaceuticals,tetramethyl-5-(1H-1,2,4-triazol-1- LP, Wilmington, ylmethyl)) DE Arsenictrioxide Trisenox Cell Therapeutic, Inc., Seattle, WA AsparaginaseElspar Merck & Co., (L-asparagine amidohydrolase, type EC-2) Inc.,Whitehouse Station, NJ BCG Live TICE BCG Organon Teknika, (lyophilizedpreparation of an attenuated Corp., Durham, strain of Mycobacteriumbovis (Bacillus NC Calmette-Gukin [BCG], substrain Montreal) bexarotenecapsules Targretin Ligand (4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-Pharmaceuticals pentamethyl-2-napthalenyl) ethenyl] benzoic acid)bexarotene gel Targretin Ligand Pharmaceuticals Bleomycin BlenoxaneBristol-Myers (cytotoxic glycopeptide antibiotics Squibb Co., NY,produced by Streptomyces verticillus; NY bleomycin A₂ and bleomycin B₂)Capecitabine Xeloda Roche (5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine) Carboplatin Paraplatin Bristol-Myers(platinum, diammine [1,1- Squibbcyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine BCNU, BiCNUBristol-Myers (1,3-bis(2-chloroethyl)-1-nitrosourea) Squibb Carmustinewith Polifeprosan 20 Implant Gliadel Wafer Guilford Pharmaceuticals,Inc., Baltimore, MD Celecoxib Celebrex Searle (as4-[5-(4-methylphenyl)-3- Pharmaceuticals,(trifluoromethyl)-1H-pyrazol-1-yl] England benzenesulfonamide)Chlorambucil Leukeran GlaxoSmithKline(4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin PlatinolBristol-Myers (PtCl₂H₆N₂) Squibb Cladribine Leustatin, 2- R. W. Johnson(2-chloro-2′-deoxy-b-D-adenosine) CdA Pharmaceutical Research Institute,Raritan, NJ Cyclophosphamide Cytoxan, Bristol-Myers(2-[bis(2-chloroethyl)amino] tetrahydro- Neosar Squibb2H-13,2-oxazaphosphorine 2-oxide monohydrate) Cytarabine Cytosar-UPharmacia & (1-b-D-Arabinofuranosylcytosine, Upjohn Company C₉H₁₃N₃O₅)cytarabine liposomal DepoCyt Skye Pharmaceuticals, Inc., San Diego, CADacarbazine DTIC-Dome Bayer AG,(5-(3,3-dimethyl-l-triazeno)-imidazole-4- Leverkusen, carboxamide(DTIC)) Germany Dactinomycin, actinomycin D Cosmegen Merck (actinomycinproduced by Streptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfaAranesp Amgen, Inc., (recombinant peptide) Thousand Oaks, CAdaunorubicin liposomal DanuoXome Nexstar((8S-cis)-8-acetyl-10-[(3-amino-2,3,6- Pharmaceuticals,trideoxy-á-L-lyxo-hexopyranosyl)oxy]- Inc., Boulder, CO7,8,9,10-tetrahydro-6,8,11-trihydroxy-1- methoxy-5,12-naphthacenedionehydrochloride) Daunorubicin HCl, daunomycin Cerubidine Wyeth Ayerst, ((1S,3 S)-3-Acetyl-1,2,3,4,6,11- Madison, NJhexahydro-3,5,12-trihydroxy-10- methoxy-6,11-dioxo-1-naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L-lyxo- hexopyranoside hydrochloride)Denileukin diftitox Ontak Seragen, Inc., (recombinant peptide)Hopkinton, MA Dexrazoxane Zinecard Pharmacia &((S)-4,4′-(1-methyl-1,2-ethanediyl)bis- Upjohn Company2,6-piperazinedione) Docetaxel Taxotere Aventis((2R,3S)-N-carboxy-3-phenylisoserine, Pharmaceuticals, N-tert-butylester, 13-ester with 5b-20- Inc., Bridgewater,epoxy-12a,4,7b,10b,13a-hexahydroxytax- NJ 11-en-9-one 4-acetate2-benzoate, trihydrate) Doxorubicin HCl Adriamycin, Pharmacia &(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a- Rubex Upjohn CompanyL-lyxo-hexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1- methoxy-5,12-naphthacenedionehydrochloride) doxorubicin Adriamycin Pharmacia & PFS Intravenous UpjohnCompany injection doxorubicin liposomal Doxil Sequus Pharmaceuticals,Inc., Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly& (17b-Hydroxy-2a-methyl-5a-androstan-3- Company, one propionate)Indianapolis, IN dromostanolone propionate Masterone Syntex, Corp.,injection Palo Alto, CA Elliott's B Solution Elliott's B Orphan Medical,Solution Inc Epirubicin Ellence Pharmacia &((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a- Upjohn CompanyL-arabino-hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedione hydrochloride) Epoetinalfa Epogen Amgen, Inc (recombinant peptide) Estramustine EmcytPharmacia & (estra-1,3,5(10)-triene-3,17- Upjohn Companydiol(17(beta))-, 3-[bis(2- chloroethyl)carbamate] 17-(dihydrogenphosphate), disodium salt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate) Etoposide phosphate Etopophos Bristol-Myers(4′-Demethylepipodophyllotoxin 9-[4,6- Squibb O-(R)-ethylidene-(beta)-D-glucopyranoside], 4′-(dihydrogen phosphate)) etoposide, VP-16 VepesidBristol-Myers (4′-demethylepipodophyllotoxin 9-[4,6-0- Squibb(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin Pharmacia& (6-methylenandrosta-1,4-diene-3, 17-dione) Upjohn Company FilgrastimNeupogen Amgen, Inc (r-metHuG-CSF) floxuridine (intraarterial) FUDRRoche (2′-deoxy-5-fluorouridine) Fludarabine Fludara Berlex (fluorinatednucleotide analog of the Laboratories, Inc., antiviral agent vidarabine,9-b-D- Cedar Knolls, NJ arabinofuranosyladenine (ara-A)) Fluorouracil,5-FU Adrucil ICN (5-fluoro-2,4(1H,3H)-pyrimidinedione) Pharmaceuticals,Inc., Humacao, Puerto Rico Fulvestrant Faslodex IPR(7-alpha-[9-(4,4,5,5,5-penta Pharmaceuticals, fluoropentylsulphinyl)nonyl]estra-1,3,5- Guayama, Puerto (10)-triene-3,17-beta-diol) RicoGemcitabine Gemzar Eli Lilly (2′-deoxy-2′,2′-difluorocytidinemonohydrochloride (b-isomer)) Gemtuzumab Ozogamicin Mylotarg WyethAyerst (anti-CD33 hP67.6) Goserelin acetate Zoladex Implant AstraZeneca(acetate salt of [D- Pharmaceuticals Ser(But)⁶,Azgly¹⁰]LHRH;pyro-Glu-His- Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro- Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄•(C₂H₄O₂)_(x) Hydroxyurea Hydrea Bristol-Myers SquibbIbritumomab Tiuxetan Zevalin Biogen IDEC, (immunoconjugate resultingfrom a Inc., Cambridge thiourea covalent bond between the MA monoclonalantibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin IdamycinPharmacia & (5,12-Naphthacenedione, 9-acetyl-7-[(3- Upjohn Companyamino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride,(7S-cis)) Ifosfamide IFEX Bristol-Myers (3-(2-chloroethyl)-2-[(2- Squibbchloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)Imatinib Mesilate Gleevec Novartis AG,(4-[(4-Methyl-1-piperazinyl)methyl]-N- Basel,[4-methyl-3-[[4-(3-pyridinyl)-2- Switzerlandpyrimidinyl]amino]-phenyl]benzamide methanesulfonate) Interferon alfa-2aRoferon-A Hoffmann-La (recombinant peptide) Roche, Inc., Nutley, NJInterferon alfa-2b Intron A Schering AG, (recombinant peptide)(Lyophilized Berlin, Germany Betaseron) Irinotecan HCl CamptosarPharmacia & ((4S)-4,11-diethyl-4-hydroxy-9-[(4- Upjohn Companypiperi-dinopiperidino)carbonyloxy]-1H- pyrano[3′,4′: 6,7]indolizino[1,2-b] quinoline-3,14(4H,12H) dione hydrochloride trihydrate)Letrozole Femara Novartis (4,4′-(1H-1,2,4-Triazol-1-ylmethylene)dibenzonitrile) Leucovorin Wellcovorin, Immunex, Corp., (L-Glutamicacid, N[4[[(2amino-5- Leucovorin Seattle, WA formyl1,4,5,6,7,8hexahydro4oxo6- pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl Ergamisol Janssen Research ((—)-(S)-2,3,5,6-tetrahydro-6-Foundation, phenylimidazo [2,1-b] thiazole Titusville, NJmonohydrochloride C₁₁H₁₂N₂S•HCl) Lomustine CeeNU Bristol-Myers(1-(2-chloro-ethyl)-3-cyclohexyl-1- Squibb nitrosourea) Meclorethamine,nitrogen mustard Mustargen Merck (2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride) Megestrol acetate Megace Bristol-Myers17α(acetyloxy)-6-methylpregna-4,6- Squibb diene-3,20-dione Melphalan,L-PAM Alkeran GlaxoSmithKline (4-[bis(2-chloroethyl) amino]-L-phenylalanine) Mercaptopurine, 6-MP Purinethol GlaxoSmithKline(1,7-dihydro-6 H-purine-6-thione monohydrate) Mesna Mesnex Asta Medica(sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate Lederle(N-[4-[[(2,4-diamino-6- Laboratoriespteridinyl)methyl]methylamino]benzoyl]- L-glutamic acid) MethoxsalenUvadex Therakos, Inc., (9-methoxy-7H-furo[3,2-g][1]- Way Exton, Pabenzopyran-7-one) Mitomycin C Mutamycin Bristol-Myers Squibb mitomycin CMitozytrex SuperGen, Inc., Dublin, CA Mitotane Lysodren Bristol-Myers(1,1-dichloro-2-(o-chlorophenyl)-2-(p- Squibb chlorophenyl) ethane)Mitoxantrone Novantrone Immunex (1,4-dihydroxy-5,8-bis[[2-[(2-Corporation hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedionedihydrochloride) Nandrolone phenpropionate Durabolin-50 Organon, Inc.,West Orange, NJ Nofetumomab Verluma Boehringer Ingelheim Pharma KG,Germany Oprelvekin Neumega Genetics Institute, (IL-11) Inc., Alexandria,VA Oxaliplatin Eloxatin Sanofi (cis-[(1R,2R)-1,2-cyclohexanediamine-Synthelabo, Inc., N,N′] [oxalato(2-)-O,O′] platinum) NY, NY PaclitaxelTAXOL Bristol-Myers (5β,20-Epoxy-1,2a,4,7β,10β,13a- Squibbhexahydroxytax-11-en-9-one 4,10- diacetate 2-benzoate 13-ester with(2R,3S)- N-benzoyl-3-phenylisoserine) Pamidronate Aredia Novartis(phosphonic acid (3-amino-1- hydroxypropylidene) bis-, disodium salt,pentahydrate, (APD)) Pegademase Adagen Enzon ((monomethoxypolyethyleneglycol (Pegademase Pharmaceuticals, succinimidyl) 11 - 17-adenosineBovine) Inc., Bridgewater, deaminase) NJ Pegaspargase Oncaspar Enzon(monomethoxypolyethylene glycol succinimidyl L-asparaginase)Pegfilgrastim Neulasta Amgen, Inc (covalent conjugate of recombinantmethionyl human G-CSF (Filgrastim) and monomethoxypolyethylene glycol)Pentostatin Nipent Parke-Davis Pharmaceutical Co., Rockville, MDPipobroman Vercyte Abbott Laboratories, Abbott Park, IL Plicamycin,Mithramycin Mithracin Pfizer, Inc., NY, (antibiotic produced byStreptomyces NY plicatus) Porfimer sodium Photofrin QLTPhototherapeutics, Inc., Vancouver, Canada Procarbazine Matulane SigmaTau (N-isopropyl-μ-(2-methylhydrazino)-p- Pharmaceuticals, toluamidemonohydrochloride) Inc., Gaithersburg, MD Quinacrine Atabrine AbbottLabs (6-chloro-9-(1-methyl-4-diethyl-amine)butylamino-2-methoxyacridine) Rasburicase Elitek Sanofi- (recombinantpeptide) Synthelabo, Inc., Rituximab Rituxan Genentech, Inc.,(recombinant anti-CD20 antibody) South San Francisco, CA SargramostimProkine Immunex Corp (recombinant peptide) Streptozocin ZanosarPharmacia & (streptozocin 2-deoxy-2- Upjohn Company[[(methylnitrosoamino)carbonyl]amino]- a(and b)-D-glucopyranose and 220mg citric acid anhydrous) Talc Sclerosol Bryan, Corp., (Mg₃Si₄O₁₀ (OH)₂)Woburn, MA Tamoxifen Nolvadex AstraZeneca((Z)2-[4-(1,2-diphenyl-1-butenyl) Pharmaceuticalsphenoxy]-N,N-dimethylethanamine 2- hydroxy-1,2,3-propanetricarboxylate(1:1)) Temozolomide Temodar Schering(3,4-dihydro-3-methyl-4-oxoimidazo[5,1- d]-as-tetrazine-8-carboxamide)Teniposide, VM-26 Vumon Bristol-Myers (4′-demethylepipodophyllotoxin9-[4,6-0- Squibb (R)-2-thenylidene-(beta)-D- glucopyranoside])Testolactone Teslac Bristol-Myers (13-hydroxy-3-oxo-13,17-secoandrosta-Squibb 1,4-dien-17-oic acid [dgr]-lactone) Thioguanine, 6-TG ThioguanineGlaxoSmithKline (2-amino-1,7-dihydro-6 H-purine-6-thione) ThiotepaThioplex Immunex (Aziridine, 1,1′,1″- Corporationphosphinothioylidynetris-, or Tris (1- aziridinyl) phosphine sulfide)Topotecan HCl Hycamtin GlaxoSmithKline ((S)-10-[(dimethylamino)methyl]-4- ethyl-4,9-dihydroxy-1H-pyrano[3′,4′: 6,7] indolizino [1,2-b]quinoline-3,14- (4H,12H)-dione monohydrochloride) Toremifene FarestonRoberts (2-(p-[(Z)-4-chloro-1,2-diphenyl-1- Pharmaceuticalbutenyl]-phenoxy)-N,N- Corp., Eatontown, dimethylethylamine citrate(1:1)) NJ Tositumomab, I 131 Tositumomab Bexxar Corixa Corp.,(recombinant murine immunotherapeutic Seattle, WA monoclonal IgG_(2a)lambda anti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab Herceptin Genentech, Inc (recombinant monoclonal IgG₁ kappaanti-HER2 antibody) Tretinoin, ATRA Vesanoid Roche (all-trans retinoicacid) Uracil Mustard Uracil Mustard Roberts Labs Capsules Valrubicin,N-trifluoroacetyladriamycin- Valstar Anthra --> 14-valerate Medeva((2S-cis)-2-[1,2,3,4,6,11-hexahydro- 2,5,12-trihydroxy-7methoxy-6,11-dioxo- [[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2- naphthacenyl]-2-oxoethylpentanoate) Vinblastine, Leurocristine Velban Eli Lilly(C₄₆H₅₆N₄O₁₀•H₂SO₄) Vincristine Oncovin Eli Lilly (C₄₆H₅₆N₄O₁₀•H₂SO₄)Vinorelbine Navelbine GlaxoSmithKline (3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate(1:2)(salt)]) Zoledronate, Zoledronic acid Zometa Novartis((1-Hydroxy-2-imidazol-1-yl- phosphonoethyl) phosphonic acidmonohydrate)

Where methods and compositions of the present invention are used fortreatment or prevention of cancer, existing therapies for the treatmentof cancer (e.g., melanoma) may be used in combination with the presentmethods.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Moles, also referred to as nevi, consist of melanocytes that have becomeactivated or transformed to proliferate and form a mass on the skin.Moles are classified as benign or dysplastic or a status between the twostates. Dysplastic moles can contain Nodal-positive melanocytes. BecauseNodal is associated with cancer stem cells, inhibition of Nodalexpression in melanocytes by Lefty was investigated. Lefty was appliedto skin to neutralize Nodal expression and render the melanocytesnonproliferative. Lefty is delivered using various delivery systems.

Similarly, other ectodermal dysplasia or carcinomas are considered themost common form(s) of cancer, which also express Nodal. Therefore,Lefty is also applied to these lesions before or after removal tomaintain homeostasis and prevent or treat cancer.

Materials and Methods:

Emollient A

-   -   Histamine dihydrochloride (0.9 mg)    -   Ethanol, USP (200 proof) (17.6 mg)    -   Trolamine, NF (9.0 mg)    -   Carbomer, NF (Carbopol 974P) (8.0 mg)    -   Methyl Paraben, NF (2.0 mg)    -   Propyl Paraben, NF (0.2 mg)    -   rhLefty (10 ng, 50 ng, 100 ng, 500 ng/ml)    -   PBS, USP Qs

Control

-   -   rhLefty (10 ng, 50 ng, 100 ng, 500 ng/ml)    -   PBS, USP

Human transformed melanocytes, transfected non-aggressive melanoma cellsand squamous cell carcinoma cells were treated with varyingconcentrations of rhLefty (10 ng-500 ng/ml) suspended in Emollient A oronly PBS (as a control) over 48 hours in vitro. Subsequently, cells werelysed and prepared for Western Blot analysis, specifically to measureNodal expression. Separately, rhLefty suspended in Emollient A, andrhLefty

suspended in PBS, were assessed by Western Blot analysis to determineLefty's protein

integrity.

-   Results: Nodal expression in human transformed melanocytes,    transfected non-aggressive melanoma cells, and squamous cell    carcinoma cells was down-regulated in a dose dependent manner in    both rhLefty/Emollient A and the rhLefty/Control.Western Blot    analysis of rhLefty/Emollient A further revealed three bands (@42    KDa, @34 KDa, @28 KDa) indicative of hLefty, indicating that Lefty    protein is not adversely affected nor degraded by Emollient A.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

I claim:
 1. A pharmaceutical composition comprising isolated Lefty,ethanol, trolamine, carbomer, methyl paraben, propyl paraben, andphosphate buffered saline (PBS); wherein said composition is formulatedas a gel for topical administration.
 2. The composition of claim 1,wherein said Lefty is a peptide.
 3. The composition of claim 1, whereinsaid Lefty is glycosylated.
 4. The composition of claim 3, wherein saidLefty is recombinant human Lefty.
 5. The composition of claim 3, whereinsaid Lefty is isolated from human embryonic stem cells.
 6. Thecomposition of claim 1, wherein said composition further compriseshistamine.
 7. The composition of claim 1, wherein said Lefty is presentin said composition as a concentration of between 1 ng/ml and 1000ng/ml.
 8. A pharmaceutical composition comprising isolated Lefty,ethanol, trolamine, carbomer, methyl paraben, propyl paraben, andphosphate buffered saline (PBS); wherein said composition is formulatedas a transdermal device for topical administration.